The objective of this proposal is to elucidate the mechanisms of anion exchange and proton-anion cotransport by the band 3 transporter of human erythrocytes. The mediated exchange of chloride and bicarbonate is essential for efficient removal of CO2 and metabolic protons from tissues and appears to be rate-limiting for CO2 excretion in the lungs during severe exercise. Cotransport of small molecules is important in epithelial membranes and volume regulation by cells. We propose 1) to characterize the net flux of proton equivalents across red cells in order to test the relationships between these two functions on band 3; 2) to characterize inhibitor interactions with the anion transporter which will test the topology of the sites and determine the conformations of the transporter that permit binding; and 3) to develop and test a new working hypothesis for the mechanisms of anion and proton transport. In the newly proposed mechanism each band 3 monomer has a single proton transport site and a single anion transport site which do not always have access to the same side of the membrane at the same time. This asynchronous behavior of these two transport sites, for which there is already evidence, results in equations for the transport velocity that exhibit 1) self-inhibition on each side of the membrane at high anion concentrations; 2) sigmoid activation kinetics for monovalent anions at low pH; 3) inhibition of proton-sulfate cotransport self-exchange at low pH; 4) the relationships between anion exchange and proton-anion (H + SO4 and H + Cl) cotransport; and 5) an explanation for residual transport after irreversible inhibition with phenylglyoxal. None of these characteristics have been so simply explained before. These studies involve measurements of net fluxes and tracer exchange across human red cell membranes, development of computer methods for non-numerical functional analysis of reaction velocities, and ligand binding to functional transmembrane proteins as probes for conformational analysis. These studies will both extend our knowledge of chloride-bicarbonate exchange and develop new analytical tools for the study of cotransport mechanisms and functional protein conformations.